Treatments of oxyhemoglobin with acetimidate results in rather specific amidination of lysine 40 alpha with a resulting increase in in oxygen affinity. This effect is most likely mediated by a displacement of the allosteric equilibrium in favor of the high affinity oxy conformation. Amidination of deoxyhemoglobin results in minimal change in oxygen affinity, presumably because of protection of lysine 40 alpha by its participation in a salt bridge. Amidination of the Beta chain causes a decrease in the effect of 2,3-diphoshoglycerate (DPG) on oxygen affinity. We are currently mapping the Beta chain to ascertain whether there is selective modification of residues at the DPG binding site. A number of imidoesters differing in size and shape have been synthesized and are being evaluated for their effectiveness in altering the functional properties of hemoglobin. Amidination of lysyl amino groups results in an increase of the pKa by 2 units. Thus, the positive charge on these groups will be maintained at higher pH in the modified protein. This has been confirmed by acid-base titration of hemoglobin and myoglobin. Both native and amidinated proteins undergo a pH dependent denaturation below pH 5. This denaturation is irreversible for the amidinated proteins, suggesting a role of unprotonated lysyl amino groups in the renaturation process.